Systemic non-nodular endosymbiotic nitrogen fixation in plants

ABSTRACT

Non-leguminous crops, e.g. wheat, maize and rice, do not form nodules and are dependant for their nutrition on fixed nitrogen from the soil, or from chemical/nitrogenous fertilizers. The present invention provides non-leguminous plants and leguminous plants, including legumes that fail to nodulate with  Rhizobia , with bacteria that enable them to fix nitrogen endophytically. Therefore, the plants contain nitrogen fixing bacteria the bacteria being located intacellularly in living plant cells.

This invention relates to nitrogen fixation and in particular, but notexclusively, to nitrogen fixation in non-leguminous and leguminousplants, to a method of establishing nitrogen fixation in non-leguminousand leguminous plants and to a plant, without nodules, obtained by wayof such a method.

Although nitrogen gas (N₂) makes up 78% of the atmosphere, it isunavailable for use by plants and most other organisms because there isa triple bond between the two nitrogen atoms, making the molecule almostinert. In order for nitrogen gas to be used for growth it must first befixed (i.e., reduced by hydrogen to ammonia) and be available in thecombined form of ammonium (NH₄ ⁺) or nitrate (NO₃ ⁻); certain types ofbacteria can carry out this biological nitrogen fixation by reducinggaseous nitrogen to ammonia (NH₃) enzymatically utilizing the enzymenitrogenase. The availability of fixed nitrogen is often the limitingfactor for plant growth and biomass production in environments wherethere is a suitable climate and availability of water to support life.

Chemically most ammonia is produced industrially by the Haber-Boschprocess by catalytically combining atmospheric nitrogen with hydrogen toform ammonia, using an iron-based catalyst at high temperature and veryhigh pressure. A relatively very small amount of ammonia is produced asa result of lightning discharges in the atmosphere.

The demand for increased crop yields in the 20th Century and into thisCentury has required biological nitrogen fixation by bacteria to besupplemented increasingly by the use of fixed nitrogen from chemicalfertilisers.

Biological nitrogen fixation can be represented by the followingequation, in which two molecules of ammonia are produced from onemolecule of nitrogen gas, at the expense of 16 molecules of adenosinetriphosphate (ATP) and a supply of electrons and protons (hydrogenions):—N₂+8H⁺+8e⁻+16ATP

2NH₃+H₂+16 ADP+16 Pi

This reaction is performed in bacteria, using an enzyme complex callednitrogenase. This enzyme consists of two proteins—an iron (Fe) proteinand a Molybdenum-Iron (Mo—Fe) protein.

The reaction occurs while N₂ is bound to the nitrogenase enzyme complex.The Fe protein is first reduced by electrons donated by ferredoxin. Thereduced Fe—protein then binds ATP and reduces the Mo—Fe protein, whichdonates electrons to N₂, producing HN═NH. In two further cycles of thisprocess (each requiring electrons donated by ferredoxin) HN═NH isreduced to H₂N—NH₂ and this in turn is reduced to 2NH₃.

Depending on the type of bacteria, the reduced ferredoxin which supplieselectrons for this process is generated by photosynthesis, respirationor fermentation.

The most familiar examples of nitrogen fixing symbioses between plantsand rhizobial bacteria are the root nodules of legumes (peas, beans,clover and the like). In these symbioses the rhizobia “invade” the plantvia root hairs or crack entry sites (i.e. intercellularly betweenepidermal cells) and cause the formation of a nodule by inducinglocalised proliferation of the plant host cells. Subsequently, therhizobia invade the cells of the nodule by penetrating the cell wall andbeing engulfed by invaginations from the plasma membrane (endocytosis).Consequently within the cells of the nodule the rhizobia are enclosed inmembrane bounded vesicles (small vacuoles) in which they fix nitrogen,utilising products of plant photosynthesis as carbon and energy sources,and supply biologically fixed nitrogen to the plant for growth anddevelopment (endosymbiosis). The bacterial microsymbiont isintracellular, but is always extra-cytoplasmic because of the integrityof the surrounding membrane.

Non-legume crops, which include the main cereals of the world e.g.wheat, maize and rice, do not form nodules and are dependent on fixednitrogen from the soil for their nutrition, or from chemical/nitrogenousfertilisers.

However, energy and environmental concerns arising from the overuse ofnitrogenous fertilisers have highlighted a need for non-leguminous cropsto obtain more of their nitrogen from the air by biological nitrogenfixation.

It is known that an intercellular, systemic, endophytic nitrogen fixinginteraction with Acetobacter diazotrophicus and Herbaspirillum spp.,without the need for nodulation, occurs naturally in Brazilian varietiesof sugar cane. Sugar cane is a member of the grass family, Gramineae,which also includes cereals. This non-nodular, intercellular, endophyticnitrogen fixing relationship may also be possible in rice, wheat, maizeand in other non-legume crops.

From several published, peer reviewed, academic papers in the art it isalso known that there is no evidence that endophytic nitrogen fixationbetween diazotrophic bacteria and the host plant occurs intracellularlyin living cells. For example, from Biological Nitrogen fixation for the21^(st) Century pp 685-692 states that there is no evidence for thepresence of endophytic diazotrophic bacteria within living cells. Jamesin “Field Crops Research 2000 pp 197-209” describes that “endophyticdiazotrophs have been observed only within intercellular spaces,vascular tissue, aerenchyma and dead cells of their hosts and not inliving cells”. Egener et al., in “MPMI Vol. 12 (1999) pp 813-819” alsodescribe that there is no evidence for endophytic diazotrophic bacteriainside living cells of plants.

The present invention aims to provide non-leguminous plants andleguminous plants, including legumes that fail to nodulate withrhizobia, with bacteria that enable them to fix nitrogen endophytically,therefore addressing many of the problems associated with the use ofchemical/nitrogenous fertilisers.

Accordingly, the present invention, in a first aspect, provides anon-leguminous or leguminous plant containing nitrogen fixing bacteria,said bacteria being located intracellularly in living plant cellsproviding fixed nitrogen to said plant.

According to the second aspect, the present invention further provides amethod of inoculating a non-leguminous or a leguminous plant withnitrogen fixing bacteria, said bacteria being located intracellularly inliving plant cells and providing fixed nitrogen to said plant.

The non-leguminous plant is preferably selected from the grass familyGramineae (includes rice [Oryza sativa], wheat [Triticum aestivum] andmaize [Zea mays]). The non-leguminous plant may also be one selectedfrom families such as: Solanaceae (includes tomato, potato and tobacco),Brassicaceae/Cruciferae (includes cabbages, turnips, oilseed rape andthe model plant Arabidopsis thaliana), Malvaceae (includes cotton),Compositae/Asteraceae (includes sunflower and lettuce), Euphorbiaceae(includes cassaya), Chenopodiaceae (includes sugar beet). The leguminousplant is preferably selected from the Leguminosae (includes soybean,clover, alfalfa, peas and other beans).

The non-leguminous plant, or leguminous plant, may be inoculated withbetween 1 to 1×10⁷ bacteria per milliliter of inoculum. Thenon-leguminous plant or leguminous plant is preferably inoculated withbetween 1 to 100 bacteria per milliliter of inoculum.

The non-leguminous plant, or leguminous plant, is more preferablyinoculated with 1-10 bacteria per millilitre of inoculum. Thenon-leguminous, or leguminous, plant is most preferably inoculated with1-2 bacteria per millilitre of inoculum. IdeallyThe non leguminous, orleguminous, plant is most preferably inoculated with one bacterium permillilitre of inoculum.

The non-leguminous, or leguminous, plant is preferably inoculated whengermination occurs or up to about seven days thereafter.

The nitrogen fixing bacterium used to inoculate the non-leguminous, orleguminous, plant is preferably Acetobacter diazotrophicus (syn.Gluconacetobacter diazotrophicus). Alternatively the nitrogen fixingbacterium used for inoculation may be a species of Herbaspirillum.

We have found that using a very low concentration of bacteria in theinoculum we can obtain plants that are healthier that those inoculatedwith higher concentrations of bacteria. We have also found thatAcetobacter diazotrophicus secretes large amounts of indole acetic acid(IAA), a plant growth hormone. It is known that the response of variousplant species to external (microbially released) IAA can vary frombeneficial to deleterious effects, depending on the concentration of IAAin the plant root. In general, when IAA is present in higherconcentrations than would normally be found in a plant, the increasedconcentration of IAA inhibits growth, and alters the phenotype of theplant. Also, at low concentrations IAA (or other plant growthsubstances) secreted by bacteria may be acting as a plant-bacterial (andother plant growth substances) signalling molecule for the intracellularendophytic establishment of Acetobacter diazotrophicus.

The nitrogen fixing bacteria may fix nitrogen in the presence of up to10% oxygen. Preferably the bacteria fix nitrogen in the presence ofbetween 2% to 7% oxygen.

The nitrogen fixing bacteria are intracellular. The intracellularnitrogen fixing bacteria are more preferably present in membrane boundedvesicles and vacuoles within the cytoplasm of the plant cell.

The nitrogen fixing bacteria are preferably found in colonies invesicles and vacuoles.

The colonies are preferably located in structures that are polyhedral inconfiguration. Most preferably the structures are substantiallyrhomboidal in shape.

These structures are capsules of levan, an oligo fructoside polymer ofβ-D-fructose secreted by A. diazotrophicus.

It is a surprising and unexpected result that the present inventionprovides a systemic, non-nodular, intracellular symbiosis between thenitrogen fixing bacteria and a non-leguminous plant, said bacteria beinglocated within the living cells of the plant. This has not been observedbefore. As mentioned previously, it is known that in other symbiosesbetween nitrogen fixing bacteria and other non-leguminous plants, e.g.,sugar cane, the bacteria exist in the intercellular spaces between cells(the apoplast) and within the dead cells of the xylem.

It is also a surprising and unexpected result that the present inventionprovides a similar systemic, non-nodular intracellular symbiosis betweennitrogen fixing bacteria and a leguminous, or non-leguminous plant, saidbacteria being located within the living cells of the plant. This hasnot been observed before.

Another surprising and unexpected result is that the nitrogen fixingbacteria, are located intracellularly in living cells within vesiclesand vacuoles in the cytoplasm in both a non-leguminous plant and aleguminous plant.

The bacteria may spread from plant cell to plant cell by division ofplant cells in the meristem and subsequent divisions thereof.

The bacteria may become systemic by moving through the xylem.Alternatively they may become systemic by division of plant cells andsubsequent divisions thereof. The bacteria may become systemic bycombinations of the above.

Accordingly, the present invention further provides, in a third aspect,a method of producing a leguminous or non-leguminous plant containingnitrogen fixing bacteria said bacteria being located intracellularly inliving plant cells, wherein said bacteria have been introduced byinoculation and have become systemic by division of plant cells andsubsequent divisions thereof.

According to a fourth aspect, the present invention still furtherprovides a leguminous or non-leguminous plant containing nitrogen fixingbacteria said bacteria being located intracellularly in living plantcells, said bacteria becoming systemic in the plant by division of plantcells and subsequent divisions thereof.

Preferably the nitrogen fixing bacteria are introduced into the plant byinoculation.

Preferably the bacteria of systemically colonized plants may bepropagated vegetatively to successive generations of non-leguminousplants or leguminous plants by vegetative propagation or by sexualpropagation of the plant.

Accordingly the present invention, in a fifth aspect, further provides amethod of producing a leguminous or non-leguminous plant containingnitrogen fixing bacteria said bacteria being located intracellularly inliving plant cells said method comprising propagating a first plantcontaining nitrogen fixing bacteria to provide successive generations ofsaid plant containing nitrogen fixing bacteria.

Preferably the nitrogen fixing bacteria are introduced into the firstplant by inoculation.

According to a sixth aspect, the present invention provides a plantcontaining nitrogen fixing bacteria said bacteria being locatedintracellularly in living plant cells, said plant, and concomitantly,said bacteria having been vegetatively propagated or sexuallypropagated. Said plant is preferably propagated from a first plantinoculated with nitrogen fixing bacteria or from progations of saidfirst plant.

According to a seventh aspect the present invention provides seedsobtainable from a plant having nitrogen fixing bacteria according to thepresent invention, said seed being such that upon germination of theseed said bacteria are located intracellularly in living cells, andprovide fixed nitrogen to said plant.

According to an eighth aspect the present invention provides a seed of aleguminous or non leguminous plant, said seed having a coat comprisingnitrogen fixing bacteria in an effective amount such that upongermination said effective amount of bacteria enter the plant and arelocated intracellularly within living cells said bacteria providingfixed nitrogen to said plant.

The seed coating will be one selected from seed coatings that are knownin the art.

The nitrogen fixing bacteria provided in the seed coat is preferablyAcetobacter diazotrophicus (syn. Gluconacetobacter diazotrophicus).Alternatively the nitrogen fixing bacterium is a species ofHerbaspirillum.

The effective amount of bacteria is between 1 to 1×10⁷ bacteria permillilitre of seed coating. The effective amount of bacteria ispreferably between 1 to 1×10⁵ bacteria per millilitre of seed coating.Most preferably the effective amount of bacteria is between 1 to 1×10³bacteria per millilitre of seed coating.

According to a ninth aspect the present invention provides a seed of aleguminous or non-leguminous plant, said seed being located in asubstrate, said substrate having nitrogen fixing bacteria in aneffective amount such that upon germination said effective amount ofbacteria enter the plant and are located intracellularly within livingcells, said bacteria providing fixed nitrogen to said plant.

The substrate is preferably a soil. However it will be appreciated thatvarious substrates for germinating seeds are known and a suitablesubstrate may be selected from those that are well known in the art.

The nitrogen fixing bacteria provided in the substrate is preferablyAcetobacter diazotrophicus (syn Gluconacetobacter diazotrophicus).Alternatively the nitrogen fixing bacterium is a species ofHerbaspirillum.

The effective amount of bacteria is between 1 to 1×10⁷ bacteria per gramof substratePreferably, the effective amount of bacteria is between 1 to1×10⁵ per gram of substrate. Most preferably the effective amount ofbacteria is between 1 to 1×10³ per gram of substrate.

The present invention will now be described, merely by way of example,with reference to the accompanying Figures, of which:—

FIGS. 1 to 13 relate to the present invention in a non-leguminous plantLycopersicon esculentum; tomato).

FIGS. 1A and B shows A. diazotrophicus UAP 5541/pRGS561 GUS invading themeristem and root hairs (A) and meristem cells (B) of lateral roots ofan inoculated plant in accordance with the present invention. Bar=25 μm(A) and 5 μm (B)

FIGS. 1C and D shows A. diazotrophicus UAP 5541/pRGS561 GUS in vesiclesand vacuoles in thin sections of cells of the meristem of an inoculatedplant in accordance with the present invention. Bar=5 μm (C and D)

FIG. 2A shows A. diazotrophicus UAP5541/pRGS561 GUS colonies in thexylem of lateral roots in an inoculated plant in accordance with thepresent invention. Bar=5 μm

FIG. 2B show A. diazotrophicus UAP5541/pRGS561 GUS in cells of thecortex near to invaded xylem in an inoculated plant in accordance withthe present invention. Bar=5 μm.

FIG. 2C shows A. diazotrophicus UAP5541/pRGS561 GUS in the in the xylemof a primary root in an inoculated plant in accordance with the presentinvention. Bar=5 μm.

FIG. 3A shows A. diazotrophicus UAP5541/pRGS561 GUS invasion of anemerging secondary lateral root of an inoculated plant in accordancewith the present invention. Bar=50 μm.

FIG. 3B shows A. diazotrophicus UAP5541/pRGS561 GUS invading an emergingsecondary lateral root via a crack entry site in an inoculated plant inaccordance with the present invention. Bar=25 μm.

FIGS. 4A and B show A. diazotrophicus UAP 5541/pRGS561 GUS colonisationof the cortex and the xylem in the root of a plant according to thepresent invention. Bar=25 μm (A and B).

FIG. 4C shows A. diazotrophicus UAP5541/pRGS561 GUS colonisation fromthe xylem to the phloem and cortex cells of a root of a plant inaccordance with the present invention. Bar=5 μm.

FIG. 4D shows colonies of A. diazotrophicus UAP5541/pRGS561 GUS inside acortex cell of a root from a plant according to the present invention.Bar=5 μm.

FIG. 5 shows A. diazotrophicus UAP5541/pRGS561 GUS colonisationspreading from the xylem to the phloem in the stem of a plant accordingto the present invention. Bar=5 μm.

FIG. 6 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) in the roottip of a plant according to the present invention. (dark fieldillumination) Bar=50 μm.

FIG. 7 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) colonisingan emerging secondary lateral root of a plant according to the presentinvention. (dark field illumination) Bar=50 μm.

FIG. 8A shows colonies of A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA)in the meristem of a root of a plant in accordance with the presentinvention. Bar=25 μm.

FIG. 8B shows colonies of A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA)in cells of the root meristem of a plant in accordance with the presentinvention. Bar=5 μm

FIG. 9 shows uniformly crystalline-like rhomboidal colonies of A.diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in cells of the root cortexin a plant according to the present invention. Bar=5 μm.

FIG. 10 shows colonies of A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA)in the vascular system of the root of a plant according to the presentinvention. (Dark field illumination) Bar=50 μm.

FIG. 11 shows colonies of A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA)in the xylem and cortex of the root of a plant according to the presentinvention. Bar=5 μm.

FIG. 12 shows the spread of A. diazotrophicus UAP 5541/pRGH562(NifH-GUSA) from the xylem to the phloem region in the stem of a plantaccording to the present invention. Bar=5 μm.

FIG. 13 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) inchloroplast containing cells in the stem of a plant according to thepresent invention. Bar=5 μm

FIGS. 14 to 19 relate to the present invention in a leguminous plant(Trifolium repens, white clover).

FIG. 14 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) in thevascular system and in cells of the cortex of the root of a plantaccording to the present invention. Bar=25 μm.

FIG. 15 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) in thexylem and phloem region of the root of a plant according to the presentinvention. Bar=5 μm.

FIG. 16 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) in thevascular system of the leaf of a plant according to the presentinvention. Bar=50 μm.

FIG. 17 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) in thexylem of a leaf vein of a plant according to the present invention.Bar=5 μm.

FIG. 18 shows spread of A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA)from the xylem to mesophyll (chloroplast containing cells) of the leafof a plant according to the present invention. Bar=25 μm.

FIG. 19 shows A. diazotrophicus UAP 5541/pRGH562 (NifH-GUSA) inchloroplast containing mesophyll cells of a leaf of a plant according tothe present invention. Bar=5 μm.

FIGS. 20 to 22 relate to the present invention in the cereal wheat(Triticum aestivum)

FIG. 20 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) invadingcells of the young root cortex of a lateral root of an inoculated plantin accordance with the present invention. Bar=25 μm

FIG. 21 shows at high magnification colonies of A. diazotrophicus, as inFIG. 20, in a cortical cell of the lateral root. Bar=5 μm

FIG. 22 shows a cluster of colonies of A. diazotrophicus UAP5541/pRGH562(NifH-GUSA) in the vacuole of a leaf epidermal cell, after treatmentwith ethanol to remove chlorophyll from the leaf. Bar=5 μm

FIGS. 23 to 25 relate to the present invention in oilseed rape (Brassicanapus)

FIG. 23 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in the xylemof the stem of an inoculated plant in accordance with the presentinvention. Bar=5 μm

FIG. 24 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in the xylemof the stem of an inoculated plant in accordance with the presentinvention. Bar=25 μm

FIG. 25 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) inchloroplast containing cells of the stem of an inoculated plant inaccordance with the present invention. Bar=5 μm

FIGS. 26 and 27 relate to the present invention in the cereal rice(Oryza sativa)

FIG. 26 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) invading acell of the root cortex of rice several cells below the epidermis of theyoung root of an inoculated plant in accordance with the presentinvention. Bar=5 μm

FIG. 27 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) invadingcells of the cortex of a mature rice root of an inoculated plant inaccordance with the present invention. Bar=5 μm

FIGS. 28 and 29 relate to the present invention in the non-legumeArabidopsis (Arabidopsis thaliana).

FIG. 28 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) invading themeristem cells of a lateral root of an inoculated plant in accordancewith the present invention. Bar=25 μm

FIG. 29 shows A. diazotrophicus UAP5541/pRGH562 (NifH-GUSA) invadingcells of the cortex of a lateral root of an inoculated plant inaccordance with the present invention. Bar=5 μm

FIG. 30 shows colonies of A. diazotrophicus within the vacuole of aliving cell of the root cortex of clover inoculated in accordance withthe present invention. Bar=25 μm

FIG. 31.A shows colonies of A. diazotrophicus at high magnificationwithin the vacuole of a living cell of the root cortex of clover.Inoculation was in accordance with the present invention. Bar=5 μm

FIG. 31.B shows another region of the root cortex of clover showingnumerous colonies of A. diazotrophicus within the vacuole of a livingcell. Bar=5 μm

FIG. 32 shows a large crystal (crystallised from water) of Levan (SigmaL8674). Bar=5 μm

SEED GERMINATION

Lycopersicon esculentum var. Ailsa Craig seeds, Trifolium repens var.Kent seeds, Triticum aestivum var. Hereford seeds, Brassica napus var.Express seeds, Oryza sativa var. Lemont seeds and Arabidopsis thalianavar. Ecotype Col-O seeds were surface sterilised in 15% (v/v)hypochlorite (Domestos) solution for fifteen minutes.

The hypochlorite solution was drained off using a sterile sieve and theseeds were subsequently rinsed six times with sterile deionised water.

The seeds were placed in a sterile 250 ml conical flask containing 15 mlof sterile deionised water. The flask was then placed in a shaker at24-26° C. in the dark and the seeds left to imbibe for three to fourdays.

The seeds were then placed on the surface of sterile agar individuallyin jars (175 ml capacity containing 50 ml of Murashige and Skoog medium(Sigma M5519), 0.8% w/v agar and 3% w/v sucrose) using sterile forceps.

Seedlings were grown for six to seven days under the followingconditions:

Temperature Day 25° C. Temperature Night 16° C. Photoperiod 0600-2200

Artificial daylight was provided by 250 μEm⁻²S⁻¹ “daylight” fluorescenttubes.

Inoculation with Acetobacter diazotrophicus:

Two strains of Acetobacter diazotrophicus were used:

-   A. diazotrophicus UAP 5541/p RGS561 (GUS)-   A. diazotrophicus UAP 5541/p RGH562 (NifH-GUSA)

Acetobacter diazotrophicus was streaked onto three 9 cm diameter Petriplates of ATGUS medium containing streptomycin 45 μg/ml and incubatedfor four to six days at 28° C.

Bacteria were scraped from the plate, using a sterile loop andtransferred to sterile 250 ml conical flasks containing 50 ml steriledeionised water. A bacterial suspension was prepared which had anoptical density of 0.5-0.6 at a wavelength of 600 nm (5×10⁸bacteria/ml). The suspension was diluted 10⁻⁹ (i.e. approx. 1bacterium/ml).

1 ml of 10⁻⁹ diluted bacterial suspension was added to the base of eachplant, after germination of said plant after six to seven days of growthin jars.

To the base of a control plant, 1 ml of sterile deionised water wasadded.

All plants were grown for a further twelve to twenty days.

Harvesting of Plants for Histochemical Staining:

The plants were removed from the agar. Excess agar was removed byblotting with paper towels. The plants were then histochemically stainedfor bacterial GUS activity; the GUS gene encodes the enzymeβ-glucoronidase, which hydrolyses X-gluc(5-bromo-4-chloro-3-indolyl-β-D-glucoronide cyclohexyl ammonium salt;Gold Biotech, USA) to form an indigo blue coloured compound.

Two controls were set up to ensure that the GUS staining reaction wasworking, the first using a sample of bacteria taken from the edge of aMurashige and Skoog 8% w/v agar plate and the second was a sample ofbacteria grown on ATGUS medium.

Method Used for Staining Bacteria for GUS Activity in Plant Tissues:

Plants previously removed from agar were placed in a vessel such that aminimal amount of staining solution is needed. The staining solutioncontaining X-gluc was added to said vessel, immersing said plants andstored in the dark overnight at 37° C. under vacuum conditions.

The plants were washed three times with 0.1 ml phosphate buffer pH7.0,and fixed with 2% (v/v) glutaraldehyde in 0.1 M phosphate buffer pH7.0The plants were subsequently viewed for staining by direct lightmicroscopic examination. Plants were dehydrated in an ethanol series andembedded in LR White Resin. Plant sections of 1 μm were prepared forviewing.

Plants inoculated with Acetobacter diazotrophicus UAP5541/pRGS561(constitutively expressing GUS) were assessed for endophyticcolonisation 12 to 20 days post inoculation. Histochemically stainedplants were examined to detect indigo blue precipitate-stainedAcetobacter diazotrophicus bacteria by direct microscopic observation ofglutaraldehyde fixed plants. For the purposes of this specificationhistochemically stained bacteria are indicated by black dots.

It was demonstrated from the results of microscopic analysis thatAcetobacter diazotrophicus inoculated at an initial concentration of 1bacterium/ml had invaded the meristematic region of lateral roots viathe root tip (FIG. 1A+B) including the meristematic cells and becomingestablished in vesicles (and then large vacuoles) in the cytoplasm ofcells of the meristem, (FIG. 1C+D). The bacteria are indicated as blackdots.

Acetobacter diazotrophicus was also seen to have invaded the xylem (FIG.2A) of the lateral roots forming colonies (indicated by black dots) andalso to have invaded cells of the cortex of the root near to the invadedxylem (FIG. 2B). The xylem of primary roots was also invaded byAcetobacter diazotrophicus (FIG. 2C).

Invasion of emerging secondary lateral roots (FIG. 3A) by crack entry(FIG. 3B) in the region of emergence was also observed. Extensivecolonisation of cortex cells also occurred (FIGS. 4A and 4B). This isprobably by spread of the bacteria (which are highly motile and known tosecrete plant cell wall degrading enzymes such as, for example,cellulases and pectinases) from young xylem elements into neighbouringcells, including the phloem (FIG. 4C). FIG. 4D shows a large cortex cellof the root of the plant colonised by Acetobacter diazotrophicus (shownas black dots).

Further analysis was carried out on tomato plants inoculated withAcetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in which theexpression of the β-glucuronidase gene (GUS) is under the control of aNifH promoter. Consequently, the bacteria will only stain blue inhistochemical analysis if nitrogenase genes are being expressed.Staining of Acetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA) asshown in FIGS. 6 to 13, was comparable to the staining of constitutivelyexpressed GUS in Acetobacter diazotrophicus UAP5541/pRGS561 GUS (FIGS. 1to 5). The bacteria in these figures are indicated by black dots.

FIG. 6 shows a plant root tip inoculated at an initial concentration of1 bacterium/ml. Bacteria can be seen as a black stain in the root tip.Bacteria also invaded emerging lateral roots (FIG. 7). FIG. 8A showsbacteria colonising meristem cells of a root and FIG. 8B is an oilimmersion picture showing black stained bacteria inside cells of themeristem. FIG. 9 shows, using an oil immersion objective lens, cellsfrom a root cortex of the plant. The bacteria (shown in black) are seeninside cells of the root cortex. It is interesting to note that thebacteria form uniform rhomboidal shaped colonies. These colonies arepackages of bacteria probably embedded in the colourless oligofructosidepolymer, levan. Acetobacter diazotrophicus is known to produce levanwhich could act to promote aggregation of bacteria into thesecrystalline-like clusters and provide thereby oxygen protection of theirnitrogenase. FIG. 10 shows the bacteria in the vascular system of theroot and FIG. 11 shows bacteria in the xylem and cells of the rootcortex. The bacteria were also found in the vascular system of the stemas shown in FIG. 12 which also shows the spread of bacteria from thexylem to the phloem region in the plant stem.

Acetobacter diazotrophicus was also seen in chloroplast containing cells(FIG. 13) in the stem of the plant.

FIGS. 14 to 19 show analysis on the legume clover plants inoculated withAcetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in which theexpression of the β-glucoronidase gene (GUS) is under the control of aNifH promoter. Consequently, the bacteria will only stain blue inhistochemical analysis if nitrogenase genes are being expressed.Intracellular invasion of living cells, systemic colonisation of theplant and staining of Acetobacter diazotrophicus UAP5541/pRGH562(NifH-GUSA) as shown in FIGS. 14 to 19 was comparable to that shown inthe non-legume, tomato plants, FIG. 6 to FIG. 13, similarly inoculatedwith Acetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA) at aninitial concentration of 1 bacterium/ml. Systemic invasion of the leaveswas very evident (FIG. 16 to FIG. 19). The bacteria in the above figuresare indicated by black dots.

FIGS. 20, 21 and 22 show analysis of cereal wheat plants inoculated withAcetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA) in which theexpression of the β-glucuronidase gene (GUS) is under the control of aNifH promoter. Consequently, the bacteria will only stain blue in thehistochemical analysis if nitrogenase genes are being expressed.Intracellular invasion of living cells, systemic colonisation of plantand staining of Acetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA)as shown on FIGS. 20 to 22 was comparable to that shown in non-legumetomato (FIGS. 6 to 13) and legume clover plants (FIGS. 14 to 19)similarly inoculated with Acetobacter diazotrophicus UAP5541/pRGH562(NifH-GUSA) at an initial concentration of 1 bacterium/ml. Systemicinvasion of the epidermal cells of the leaves was very evident (FIG.22). The bacteria in the above figures are indicated by black dots.

FIGS. 23, 24 and 25 show analysis on oilseed rape plants similarlyinoculated with Acetobacter diazotrophicus UAP5541/pRGH562 (NifH-GUSA).Intracellular invasion of living cells, systemic colonization of theplant and the staining of Acetobacter diazotrophicus UAP5541/pRGH562(NifH-GUSA) was comparable to that observed in tomato, clover and wheatsimilarly inoculated.

FIGS. 26 and 27 show analysis of the cerial rice (Oryza sativa)similarly inoculated with Acetobacter diazotrophicus UAP 5541/pRGH 562(NifH-GUSA). Intracellular invasion of living cells, systemiccolonisation of the plant and the staining of Acetobacter diazotrophicusUAP 5541/pRGH 562 (Nif H-GUSA) was comparable to that observed intomato, clover, wheat and oilseed rape plants similarly inoculated.

FIGS. 28 and 29 show analysis of the model plant Arabidopsis thalianainoculated with Acetobacter diazotrophicus UAP 5541/pRGH562 (Nif H-GUSA)in accordance with the present invention. Intracellular invasion ofliving cell, systemic colonisation of the plant and the staining ofAcetobacter diazotrophicus UAP 5541/pRGH562 (Nif H-GUSA) was comparableto that observed in tomato, clover, wheat, oilseed rape and cereal riceplants similarly inoculated.

Method Used for Staining with Neutral Red

Neutral Red (Merck index No. 6571) is a biological stain which isnon-toxic. Plant cells are still viable after staining with 0.01% W/VNeutral Red in water. Plants inoculated with Acetobacter diazotrophicusin accordance with the present invention were placed in a solution ofneutral red (0.9% W/V in water) for 30 minutes. The plants were thenwashed and prepared for microscopic examination.

FIG. 30, 31A and 31B show analysis of Acetobacter diazotrophicus inliving cells of clover. This analysis was performed to ensure that theAcetobacter diazotrophicus bacteria inoculated into clover in accordancewith the present invention were actually present intracellularly inliving cells. Neutral red was used for this purpose. At low pH (a pHless than 7.0) cellular compartments that are acidic stain red. Athigher pH (a pH above 7.0) neutral red is presented as a yellow stain.FIGS. 30 and 31A show colonies (black dots) of Acetobacterdiazotrophicus within a vacuole of a living cell of the root cortex ofclover inoculated in accordance with the present invention. FIG. 31Bshows another region of the root cortex of clover with Acetobacterdiazotrophicus (black dots) present in the vacuole of a living cell.

Acetobacter diazotrophicus is present in living cells as polyhedralcolonies. These polyhedral structures are caused by the secretion of apolymer of β-D-fructose called Levan. Crystals of Levan isolated fromErwinia herbicola (sigma cat. No. L8647) closely resemble the shape ofcolonies of Acetobacter diazotrophicus found in plants inoculated inaccordance with the present invention (FIG. 32).

Wheat (Triticum aestivum) and clover (Trifolium repens), innoculatedwith A. diazotrophicus UAP 5541/pRGH562 (Nif H-GUSA) in accordance withthe present invention, were transferred after two weeks inoculation injars (75 ml capacity containing 50 ml of Murashige and Skoog medium,0.8% W/V agar and 3% W/V sucrose), to (seed and cutting) compost in potsfor four weeks. The plants in pots were incubated under clean growthroom conditions (25° C. day temperature, 16° C. night temperature,photoperiod of 250 μEm⁻²S⁻¹ from ‘Daylight’ fluorescent tubes,0600-2200) and watered with sterile water. Plants were assayed fornitrogenase activity using the acetylene reduction assay. Uninoculatedcontrols were also similarly transferred from jars to compost andassayed for nitrogenase activity using the acetylene reduction assay.

Acetylene Reduction Assay

Nitrogenase, the enzyme responsible for the reduction of gaseousnitrogen (N≡N) to ammonia (NH₃) (nitrogen fixation), was assayed by gaschromatography. In this assay, plants are incubated with excessacetylene gas (H—C≡CH) which is reduced by nitrogenase acting on thetriple bond of acetylene to yield ethylene (H2—C═C—H₂). Plants wererinsed in sterile water and transferred to 75 ml Pyrex tubes which werethen capped with gas tight Subaseals™. 10% of the air volume was removedusing a hypodermic syringe, and replaced with acetylene. The sampleswere returned to the growth room and incubated for 24 hours under thesame conditions used for the growth of plants inoculated with A.diazotrophicus (25° C., day temperature, 16° C., night temperature,photoperiod of 250 μEm⁻²S⁻¹ from “Daylight” fluorescent tubes,0600-2200). Samples of gases (0.5 ml) were removed in syringes andanalysed for ethylene production with a Pye Unicam PU 4500 gaschromatograph with 183 cm (2.0 mm internal diameter) glass columncontaining ‘Propack N’ with a mesh size of 80-100. The mobile phasecarrier was N₂ at a flow rate of 27 ml min⁻¹. The oven containing thecolumn was set at 60° C. and the flame detector set to 121° C. Theinstrument was calibrated (peak height: ethylene (number of nanomoles)per 0.5 ml sample) using a standard curve.

Uninoculated Inoculated (control) WHEAT (nanomoles ethylene per 24hours) 31* 7 12* 6 6 7 6 7 15* 6 *Nitrogenase activity of individualwheat plant (-control) 24, 6 & 9 nanomoles ethylene respectively. CLOVER(nanomoles ethylene per 24 hours) 4 6 6 7 5 5 3 3 5 6 15* 3 *Nitrogenaseactivity of individual clover plant (-control): 12, nanomoles ethylene.

When clover was inoculated with Rhizobium leguminosarum biovar Trifolii(RCR5), under these growth conditions, nodulated plants were producedand these had a mean nitrogenase activity per clover plant of 60nanomoles ethylene per 24 hours.

1. A non-leguminous or leguminous plant containing nitrogen fixingbacteria, Acetobacter diazotrophicus (syn. Gluconacebobacterdiazotrophicus), said bacteria being located intracellularly in livingplant cells of said plant and providing fixed nitrogen to said plantwherein said nitrogen fixing bacteria are present in membrane boundvescicles and vacuoles within the cytoplasm of the living plant cells.2. A plant according to claim 1 obtained by a method of inoculating saidplant with between 1 and 100 of said bacteria per millilitre ofinoculum.
 3. A plant according to claim 1 obtained by a method ofinoculating said plant with between 1 and 10 of said bacteria permillilitre of inoculum.
 4. A plant according claim 1 wherein thebacteria spread from plant cell to plant cell by division of plant cellsin the meristem and subsequent divisions thereof.
 5. A plant accordingto claim 1 wherein the bacteria become systemic by moving through thexylem.
 6. A plant according to claim 1 wherein the bacteria becomesystemic by divisions of plant cells and subsequent divisions thereof.7. A method of producing a leguminous or non-leguminous plant inaccordance with claim 1, wherein said bacteria are introduced byinoculation of said plant with between 1 and 100 bacteria per millilitreof inoculum when germination of said plant occurs or up to seven daysthereafter, and wherein said bacteria become systemic by division ofplant cells and subsequent divisions thereof.
 8. A method of inoculatinga non-leguminous or a leguminous plant with nitrogen fixing bacteria,Acetobacter diazotrophicus (syn. Gluconacetobacter diazotorphicus toproduce a plant in accordance with claim 1 which comprises inoculatingsaid plant with between 1 and 100 of said bacteria per millilitre ofinoculum when germination of said plant occurs or up to seven daysthereafter.
 9. A method according to claim 8 wherein the non-leguminousor leguminous plant is inoculated with 1-10 bacteria per millilitre ofinoculum.